Characterization of different aspects of Mycobacterium tuberculosis physiology: impact on drug discovery and virulence

Mycobacterium tuberculosis is a remarkably successful human pathogen, which causes nearly two million death annually. Although tuberculosis has been widely studied for more than a century, the mechanisms by which it causes the disease, are poorly understood. Recent progress of molecular genetics beside of completion of its genome sequence, have been providing tools for new approaches to a better understanding of M.tuberculosis biochemistry, physiology and pathogenesis. Here we show a series of studies in which, taking advantage of a genetic approach, we characterized different aspects of M. tuberculosis physiology which might have a strong impact in drug discovery and in the understanding of its virulence mechanism. Producing a knock-down mutant of sigA, encoding the principal sigma factor of M. tuberculosis, we showed that it is essential and posed the basis for the development of a simple in vitro test to select molecules active on dormant bacteria. Similar results were obtained using .a genetic system in which the expression of different toxins belonging to toxin-antitoxin modules can be induced. We also produced an M. tuberculosis mutant in which the gene encoding the alternative sigma factor SigE and its cognate anti-sigma factor RseA were deleted. The SigE-RseA regulatory network is extremely important to stop phagosome maturation and consequently for virulence. This mutant strain will be complemented with the sigE-rseA couple carrying different mutations that will allow the further characterization of this regulatory network. Finally, we also characterized the regulation of the expression of the PE_PGRS30 structural gene and the role of its PGRS domain in virulence. Keywords: M.tuberculosis, sigA, sigE, Toxin-antitoxin, PE_PGRSMycobacterium tuberculosis is a remarkably successful human pathogen, which causes nearly two million death annually. Although tuberculosis has been widely studied for more than a century, the mechanisms by which it causes the disease, are poorly understood. Recent progress of molecular genetics beside of completion of its genome sequence, have been providing tools for new approaches to a better understanding of M.tuberculosis biochemistry, physiology and pathogenesis. Here we show a series of studies in which, taking advantage of a genetic approach, we characterized different aspects of M. tuberculosis physiology which might have a strong impact in drug discovery and in the understanding of its virulence mechanism. Producing a knock-down mutant of sigA, encoding the principal sigma factor of M. tuberculosis, we showed that it is essential and posed the basis for the development of a simple in vitro test to select molecules active on dormant bacteria. Similar results were obtained using .a genetic system in which the expression of different toxins belonging to toxin-antitoxin modules can be induced. We also produced an M. tuberculosis mutant in which the gene encoding the alternative sigma factor SigE and its cognate anti-sigma factor RseA were deleted. The SigE-RseA regulatory network is extremely important to stop phagosome maturation and consequently for virulence. This mutant strain will be complemented with the sigE-rseA couple carrying different mutations that will allow the further characterization of this regulatory network. Finally, we also characterized the regulation of the expression of the PE_PGRS30 structural gene and the role of its PGRS domain in virulence. Keywords: M.tuberculosis, sigA, sigE, Toxin-antitoxin, PE_PGR

Using Waste Ceramic Dust in Stabilization of Clay Soils

A considerable amount of the globally produced ceramic goes to waste daily. Ceramic wastes that are sent to the landfills have negative effects on soil, water, and the environment, as they contain aluminum, silica and iron oxide. The waste water leaching to the soil contains insoluble particular matter or heavy metals and could damage the plants. The air emission of the ceramic waste dumping process is also very high and it has negative effect on environment and human health by spreading dust. Using waste ceramic dust in soil stabilization could be for better disposal of such waste. The usage of natural resources can be minimized as well as prevent additional environmental burden and emissions. For this purpose, three local clay soil samples from Erbil in North-Iraq were gathered and the effects of addition of waste ceramic dust on mechanical properties of these samples were experimentally examined in two different grading sizes. The ceramic dust with particle sizes passing sieve No.40 and sieve No.10, in the proportion of 0, 5, and 10% percentages were used. The study showed that with an increase in ceramic dust percentages, liquid limit, plastic limit, plasticity index, optimum moisture content of the clay decreased. On the other hand maximum dry density, unconfined compressive strength and California bearing ratio increased. The study showed that addition of No.10 gradation ceramic dust results in higher improvement compared to the same amount of ceramic dust in No.40 size. The current work concludes that soil stabilized with the right type and ratio of ceramic dust could be suitable for a sustainable highway construction subgrade by reducing the design thickness and potentially be more economi

Evaluating the role of vitamin D receptor polymorphisms on susceptibility to tuberculosis among Iranian patients: a case-control study

receptor polymorphisms o

14-3-3 Activated Bacterial Exotoxins AexT and ExoT Share Actin and the SH2 Domains of CRK Proteins as Targets for ADP-Ribosylation

Bacterial exotoxins with ADP-ribosyltransferase activity can be divided into distinct clades based on their domain organization. Exotoxins from several clades are known to modify actin at Arg177; but of the 14-3-3 dependent exotoxins only Aeromonas salmonicida exoenzyme T (AexT) has been reported to ADP-ribosylate actin. Given the extensive similarity among the 14-3-3 dependent exotoxins, we initiated a structural and biochemical comparison of these proteins. Structural modeling of AexT indicated a target binding site that shared homology with Pseudomonas aeruginosa Exoenzyme T (ExoT) but not with Exoenzyme S (ExoS). Biochemical analyses confirmed that the catalytic activities of both exotoxins were stimulated by agmatine, indicating that they ADP-ribosylate arginine residues in their targets. Side-by-side comparison of target protein modification showed that AexT had activity toward the SH2 domain of the Crk-like protein (CRKL), a known target for ExoT. We found that both AexT and ExoT ADP-ribosylated actin and in both cases, the modification compromised actin polymerization. Our results indicate that AexT and ExoT are functional homologs that affect cytoskeletal integrity via actin and signaling pathways to the cytoskeleton

Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE

Abstract σE is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized. Several sigma factors are post-translationally regulated by proteins named anti-sigma factors, which prevent their binding to RNA polymerase. Rv1222 (RseA), whose gene lays immediately downstream sigE, has been proposed in the past as the σE-specific anti sigma factor. However, its role as anti-sigma factor was recently challenged and a new mechanism of action was hypothesized predicting RseA binding to RNA polymerase and DNA to slow down RNA transcription in a not specific way. In this manuscript, using specific M. tuberculosis mutants, we showed that by changing the levels of RseA expression, M. tuberculosis growth rate does not change (as hypothesized in case of non-specific decrease of RNA transcription) and has an impact only on the transcription level of genes whose transcriptional control is under σE, supporting a direct role of RseA as a specific anti-σE factor

Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system

Abstract Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for Mycobacterium tuberculosis based on two different repressors (TetR/Pip-OFF system). This system, causing the repression of the target gene in response to anhydrotetracycline (ATc), has been successfully used in several laboratories to characterize essential genes in different mycobacterial species both in vitro and in vivo. One of the limits of this system was its instability, leading to the selection of mutants in which the expression of the target gene was no longer repressible. In this paper we demonstrated that the instability was mainly due either to the loss of the integrative plasmid carrying the genes encoding the two repressors, or to the selection of a frameshift mutation in the gene encoding the repressors Pip. To solve these problems, we (i) constructed a new integrative vector in which the gene encoding the integrase was deleted to increase its stability, and (ii) developed a new integrative vector carrying the gene encoding Pip to introduce a second copy of this gene in the chromosome. The use of these new tools was shown to reduce drastically the selection of escape mutants

Mycobacterium tuberculosis Rv3160c is a TetR-like transcriptional repressor that regulates expression of the putative oxygenase Rv3161c

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a major health threat listed among the top 10 causes of death worldwide. Treatment of multidrug-resistant Mtb requires use of additional second-line drugs that prolong the treatment process and result in higher death rates. Our team previously identified a 2-pyridone molecule (C10) that blocks tolerance to the first-line drug isoniazid at C10 concentrations that do not inhibit bacterial growth. Here, we discovered that the genes rv3160c and rv3161c are highly induced by C10, which led us to investigate them as potential targets. We show that Rv3160c acts as a TetR-like transcriptional repressor binding to a palindromic sequence located in the rv3161c promoter. We also demonstrate that C10 interacts with Rv3160c, inhibiting its binding to DNA. We deleted the rv3161c gene, coding for a putative oxygenase, to investigate its role in drug and stress sensitivity as well as C10 activity. This Δrv3161c strain was more tolerant to isoniazid and lysozyme than wild type Mtb. However, this tolerance could still be blocked by C10, suggesting that C10 functions independently of Rv3161c to influence isoniazid and lysozyme sensitivity

NUDT6 and NUDT9, two mitochondrial members of the NUDIX family, have distinct hydrolysis activities

International audienceThe 22 members of the NUDIX (NUcleoside DIphosphate linked to another moiety, X) hydrolase superfamily can hydrolyze a variety of phosphorylated molecules including (d)NTPs and their oxidized forms, nucleotide sugars, capped mRNAs and dinucleotide coenzymes such as NADH and FADH. Beside this broad range of enzymatic substrates, the NUDIX proteins can also be found in different cellular compartments, mainly in the nucleus and in the cytosol, but also in the peroxisome and in the mitochondria. Here we studied two members of the family, NUDT6 and NUDT9. We showed that NUDT6 is expressed in human cells and localizes exclusively to mitochondria and we confirmed that NUDT9 has a mitochondrial localization. To elucidate their potential role within this organelle, we investigated the functional consequences at the mitochondrial level of NUDT6-and NUDT9deficiency and found that the depletion of either of the two proteins results in an increased activity of the respiratory chain and an alteration of the mitochondrial respiratory chain complexes expression. We demonstrated that NUDT6 and NUDT9 have distinct substrate specificity in vitro, which is dependent on the cofactor used. They can both hydrolyze a large range of low molecular weight compounds such as NAD + (H), FAD and ADPR, but NUDT6 is mainly active towards NADH, while NUDT9 displays a higher activity towards ADPR

Differential polar localization between PE_PGRS30 and PE_PGRS33 spatial distribution in <i>Mtb</i>.

<p>A) Schematic representation of the PE_PGRS33-derived chimeras expressed in <i>Mtb</i> H37Rv. B) Confocal images of <i>Mtb</i> H37Rv expressing PE_PGRS33^GFP and ₃₃PE^GFP using a 63× objective. In the inbox, a 100× image obtained overlapping green channel and transmission image is shown. Sample line profile and ratio between the GFP emission intensity at the bacterium pole and the GFP emission intensity in the cytoplasm between PE_PGRS30^GFP and PE_PGRS33^GFP (C) and between ₃₀PE^GFP and ₃₃PE^GFP (D). Twenty R values were analyzed for each <i>Mtb</i> strain under study. Two-tailed Student's <i>t</i>-test was used to analyze R ratio (<b>*</b><i>p</i><0.05, <b>**</b><i>p<</i>0.01).</p

Scheme showing the constructs expressing PE_PGRS30 used in this study.

<p>Schematic representation of native full length PE_PGRS30 gene with the indication of the different protein domains (A). List of constructs generated in pMV206 and expressing the PE_PGRS30 functional chimeras, tagged with green fluorescent protein (B). The constructs were transformed in <i>Mtb</i> H37Rv, <i>M. smegmatis</i> and <i>M. bovis</i> BCG.</p